DEV011767 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Cell survival in serum-free medium culture condition. (A) Cell survival in the absence of serum was determined by flow cytometry measuring dead cells via 7AAD incorporation. The percentage of live cells (7AAD⁻ cells) at days 1, 2 and 3 of culture is shown in the left panel, whereas the right panel shows the absolute number of live cells recovered from 5 ml of culture. Data are presented as the mean ±s.e.m. (n=5). Serum-supplemented culture was used as a positive control of cell survival. (B) Absolute live cell number recovered from day-3 EBs derived from control Bry-GFP ES cells or from Bcl2-overexpressing Bry-GFP ES cells, stimulated or not with 4 ng/ml Bmp4. Data are presented as the mean ±s.e.m. (n=3). (C) GFP expression profiles of day-3 EBs overexpressing Bcl2, cultured with (black line) or without (red line) Bmp4. Bcl2-IRES-puro was expressed under the control of the chicken β-actin promoter. After electroporation into Bry-GFP ES cells, puromycin-resistant ES clones were amplified and tested for Bcl2 overexpression. EBs were generated as described in Material and methods, either from individual clones or from pools of clones.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Quantification of Rex1 and Fgf5 expression levels by real-time PCR. RNA was purified from ES cells and from day 1, 2 and 3 EBs grown in the serum-free condition. (A) The expression levels for β-actin, Fgf5 and Rex1 genes were measured from reverse-transcribed cDNA and the ΔCt was calculated between the genes of interest and β-actin for each sample. Data are presented as mean value of triplicate well ±s.e.m. (B) Fold changes were calculated using relative level of expression in ES cells set at 1. Real-time PCR analysis was performed using the Exiqon Universal ProbeLibrary and Assay Design for PCR primer (Roche). Data shown are representative of three independent experiments.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Induction of mesoderm differentiation is highly efficient only in the presence of Bmp4 or serum. Flow cytometric analysis of GFP+ cells in day-3.5 EBs grown in serum-free culture conditions supplemented with the indicated cytokine at day 0. Bmp4 was added at 4 ng/ml.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Bry-GFP expression level is not affected by Bmp4 concentration. (A) GFP expression profiles of day-3.5 and day-4 EBs stimulated with serum (black line) or with the indicated amount of Bmp4 (red line). (B) Total GPF+ cell recovery from EBs derived from serum-free culture grown with or without Bmp4. EBs were grown for up to 5 days in serum-free culture without added factors (black bars), with 4 ng/ml Bmp4 (white bars) or 20 ng/ml Bmp4 (gray bars). At the indicated day, a 5-ml culture of EBs was harvested, the total number of live cells counted and the percentage of GFP+ live cells was measured by flow cytometry. *A statistically significant difference (P<0.05) compared with the day-2 cell counts. Data are presented as mean value ±s.e.m. (n=5).

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Absence of visceral endoderm on developing EBs. (A) E7.25 embryo sections were stained with Hematoxylin and Eosin (H&E) (left panel) or with Fluorescein DBA (middle panel) to mark the visceral endoderm layer (right panel, bright field of the DBA-stained section). (B) H&E staining of day-3 EB section grown in serum-free culture condition with 4 ng/ml Bmp4. EBs day 2 (C) and day 3 (D) stained with Fluorescein DBA (lower panels) showing the absence of visceral endoderm-like outer layer. Photos are 10× magnification, Olympus BX51 microscope, Colorview12 camera. Right-hand photo in B is 40×. Paraffin sections of EBs or E7.25 embryos within decidual tissue were stained with H&E or with Fluorescein Dolichos biflorus agglutinin (Vector Laboratories) as described in Soudais et al. (1995).

  Reference

  Soudais, C., Bielinska, M., Heikinheimo, M., MacArthur, C. A., Narita, N., Saffitz, J. E., Simon, M. C., Leiden, J. M. and Wilson, D. B. (1995). Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro. Development 121, 3877-3888.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S6. Hematopoietic potential of individual blast colonies grown from hemangioblast-derived EBs cultured in serum-free conditions in the presence of Bmp4, activin A and Fgf. Hematopoietic potential was tested for individual blast colonies (five colonies, B1 to B5, are shown here) in secondary replating after 4 days of expansion as described previously (Choi et al., 1998).

• Supplemental Figure S7 (Adobe PDF) -

  Fig. S7. The RI and F1 ES cell lines efficiently form hematopoietic precursors upon stimulation with Bmp4 followed by activin A, bFGF and VEGF in serum-free culture condition. (A) F1 and RI ES cells were induced to differentiate in serum-free culture conditions with Bmp4, followed by activin A, bFGF and VEGF at day 2.5. At day 5 of EB differentiation, cells were assayed for hematopoietic progenitors by colony-forming ability in the secondary replating assay. *A statistically significant difference (P<0.05) compared with the respective serum controls. Data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m. (B) The relative expression of CD41 and CD34 was determined by flow cytometric analysis on cells derived from day-4 and day-5 EBs grown in serum-free culture stimulated with the BAFV mix or with serum. Data are shown for the F1 ES cells. B, Bmp4; A, activin A; F, bFGF; V, VEGF.